Two formulations of pneumococcal vaccines are currently available to prevent invasive disease in adults and children. However, these vaccines will not protect against the majority of Streptococcus pneumoniae serotypes. The use of highly conserved cell-wall-associated proteins in vaccines may circumvent this problem. A proteomics approach was used to identify 270 S. pneumoniae cell-wall-associated proteins, which were then screened in a process that included in-silico, in-vitro and in-vivo validation criteria. Five potential candidates for inclusion in a vaccine were selected, expressed in Escherichia coli, and purified for use in immunisation experiments. These proteins were detected in at least 40 different serotypes of S. pneumoniae, and were expressed in S. pneumoniae isolates causing infection. Two of the five candidate proteins, the putative lipoate protein ligase (Lpl) and the ClpP protease, resulted in a reduced CFU titre and a trend towards reduced mortality in an animal sepsis model for investigating new S. pneumoniae protein vaccines.